9NVU

Engineered OrufIscB-omegaRNA-target DNA complex

Summary for 9NVU

• Entry DOI: 10.2210/pdb9nvu/pdb
• EMDB information: 49856
• Descriptor: NTS, OrufIscB-REC-swap 49, DNA TS, ... (6 entities in total)
• Functional Keywords: gene editing, iscb, omega, rna-guided nuclease, rna binding protein-rna-dna complex, rna binding protein/rna/dna
• Biological source: synthetic construct; More
• Total number of polymer chains: 4
• Total formula weight: 163463.34

Authors

Xu, P.,Zhu, S.,Zhang, F. (deposition date: 2025-03-21, release date: 2025-05-21)

Primary citation

Kannan, S.,Altae-Tran, H.,Zhu, S.,Xu, P.,Strebinger, D.,Oshiro, R.,Faure, G.,Moeller, L.,Pham, J.,Mears, K.S.,Ni, H.M.,Macrae, R.K.,Zhang, F.
Evolution-guided protein design of IscB for persistent epigenome editing in vivo.
Nat.Biotechnol., 2025

PubMed Abstract: Naturally existing enzymes have been adapted for a variety of molecular technologies, with enhancements or modifications to the enzymes introduced to improve the desired function; however, it is difficult to engineer variants with enhanced activity while maintaining specificity. Here we engineer the compact Obligate Mobile Element Guided Activity (OMEGA) RNA-guided endonuclease IscB and its guiding RNA (ωRNA) by combining ortholog screening, structure-guided protein domain design and RNA engineering, and deep learning-based structure prediction to generate an improved variant, NovaIscB. We show that the compact NovaIscB achieves up to 40% indel activity (~100-fold improvement over wild-type OgeuIscB) on the human genome with improved specificity relative to existing IscBs. We further show that NovaIscB can be fused with a methyltransferase to create a programmable transcriptional repressor, OMEGAoff, that is compact enough to be packaged in a single adeno-associated virus vector for persistent in vivo gene repression. This study highlights the power of combining natural diversity with protein engineering to design enhanced enzymes for molecular biology applications.

PubMed: 40335752
DOI: 10.1038/s41587-025-02655-3

Experimental method

ELECTRON MICROSCOPY (2.71 Å)